177 T-cell vaccine design for Streptococcus pneumoniae: an in silico approach

Streptococcus pneumoniae (pneumococcus) remains an important cause of meningitis, bacteremia, acute otitis media, community acquired pneumonia associated with significant morbidity, and mortality world wide. Conjugated polysaccharide, glycoconjugated, and capsular polysaccharide based vaccines were existent for pneumococcal disease but are still specific and restricted to serotypes of S. pneumoniae. Proteome of eight serotypes of S. pneumoniae was retrieved and identified in common proteins (Munikumar et al., 2012). 18 membrane proteins were distinguished from 1657 common proteins of eight serotypes of S. pneumoniae. Implementing comparative genomic approach and subtractive genomic approach, three membrane proteins were predicted as essential for bacterial survival and non-homologous to human (Munikumar et al., 2012; Umamaheswari et al., 2011). ProPred server was used to propose four promiscuous T-cell epitopes from three membrane proteins and validated through published positive control, SYFPEITHI and immune epitope database (Munikumar et al., in press). The four epitopes docked into peptide binding region of predominant HLA-DRB alleles with good binding affinity in Maestro v9.2. The T-cell epitope 89-VVYLLPILI-97 and HLA-DRB5^?0101 docking complex was with best XPG score (?13.143?kcal/mol). Further, the stability of the complex was checked through molecular dynamics simulations in Desmond v3.3. The simulation results had revealed that the complex was stable throughout 5000?ps (Munikumar et al., in press). Thus, the epitope would be the ideal candidate for T-cell driven subunit vaccine design against selected serotypes of S. pneumoniae.     

161 Discovery of potent KdsA inhibitors of Leptospira interrogans through homology modeling,docking, and molecular dynamics simulations

Leptospira interrogans is the foremost cause of human leptospirosis. Discovery of novel lead molecules for common drug targets of more than 250 Leptospira serovars is of significant research interest. Lipopolysaccharide (LPS) layer prevent entry of hydrophobic agents into the cell and protect structural integrity of the bacterium. KDO-8-phosphate synthase (KdsA) catalyzes the first step of KDO biosynthesis that leads to formation of inner core of LPS. KdsA was identified as a potential drug target against Leptospira interrogans through subtractive genomic approach, metabolic pathway analysis, and comparative analysis (Amineni et al., 2010). The present study rationalizes a systematic implementation of homology modeling, docking, and molecular dynamics simulations to discover potent KdsA inhibitors (Pradhan et al., 2013; Umamaheswari et al., 2010). A reliable tertiary structure of KdsA in complex with substrate PEP was constructed based on co-crystal structure of Aquifex aeolicus KdsA synthase with PEP using Modeller9v10. Geometry-based analog search for PEP was performed from LigandInfo database to generate an in house library of 352 ligands. The ligand data-set was docked into KdsA active site through three-stage docking technique (HTVS, SP, and XP) using Glidev5.7. Thirteen lead molecules were found to have better binding affinity compared to PEP (XP Gscore?=??7.38?kcal/mol; Figure 1). The best lead molecule (KdsA- lead1 docking complex) showed XP Gscore of ?10.26?kcal/mol and the binding interactions (Figure 2) were correlated favorably with PEP–KdsA interactions (Figure 1). Molecular dynamics simulations of KdsA– lead1 docking complex for 10?ns had revealed that the complex (Figure 3) remained stable in closer to physiological environmental condition. The predicted pharmacological properties of lead1 were well within the range of a drug molecule with good ADME profile, hence, would be intriguing towards development of potent inhibitor molecule against KdsA of Leptospira.     

Improvement of the crystallizability and expression of an RNA crystallization chaperone

Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNA-Fab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of ΔC209 P4-P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of Fab-RNA complex.     

Effect of structurally constrained oxime-ether linker on PPAR subtype selectivity: Discovery of a novel and potent series of PPAR-pan agonists

A novel series of thaizole and oxazole containing phenoxy acetic acid derivatives is reported as PPAR-pan agonists. Incorporation of structurally constrained oxime-ether based linker in the chemotype of a potent PPARδ selective agonist GW-501516 was adapted as designing strategy. In vitro, selected test compounds 12a, 12c, 17a and 18a showed PPAR-pan agonists activities and among these four compounds tested, 12a emerged as highly potent and efficacious compound, while 17a exhibited moderate and balanced PPAR-pan agonistic activity. In vivo, selected test compounds 12a and 17a exhibited significant anti-hyperglycemic and anti-hyperlipidemic activities in relevant animal models. These results support our hypothesis that the introduction of structurally constrained oxime-ether linker between lipophilic tail and acidic head plays an important role in modulating subtype selectivity and subsequently led to the discovery of potent PPAR-pan agonists.     

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Abstract

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K_a) of methotrexate to muscle cystatin was found to be 1?×?10^?7 M^?1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.

Communicated by Ramaswamy H. Sarma     

Functional characterization of coat protein and V2 involved in cell to cell movement of Cotton leaf curl Kokhran virus-Dabawali

The functional attributes of coat protein (CP) and V2 of the monopartite begomovirus, Cotton leaf curl Kokhran virus- Dabawali were analyzed in vitro and in vivo by their overexpression in E. coli, insect cells and transient expression in the plant system. Purified recombinant V2 and CP proteins were shown to interact with each other using ELISA and surface plasmon resonance. Confocal microscopy of Sf21 cells expressing V2 and CP proteins revealed that V2 localized to the cell periphery and CP to the nucleus. Deletion of the N terminal nuclear localization signal of CP restricted its distribution to the cytoplasm. GFP-V2 and YFP-CP transiently expressed in N. benthamiana plants by agroinfiltration substantiated the localization of V2 to the cell periphery and CP predominantly to the nucleus. Interestingly, upon coinfiltration, CP was found both in the nucleus and in the cytoplasm along with V2. These results suggest that the interaction of V2 and CP may have important implications in the cell to cell movement.     

Aggregation and inactivation of pancreatic cystatin by riboflavin-derived singlet oxygen and flavin triplet state: polyphenols as preventive agents

Caprine pancreatic thiol proteinase inhibitor (PTPI) a cystatin superfamily variant has high affinity for cysteine proteinases providing tight regulation of their proteolytic potential. Oxidative stress buildup in various pancreatic pathologies worsens the disease course often by disturbing the delicate balance between proteinases and their inhibitors. We aimed to study the effect of reactive oxygen species (ROS) on PTPI and to determine the potency of caffeic acid, curcumin and quercetin as agents against the inflicted damage. Fluorescence spectroscopy, polyacrylamide gel electrophoresis, and papain inhibitory assay revealed that photoilluminated riboflavin severely challenged the functional and structural integrity of the inhibitor. Three hundred and fifty micromolar affeic acid or quercetin prevented the damage. Curcumin, however, failed to reverse the changes completely. Conclusively, PTPI rendered dysfunctional by ROS may explain the increased necrotic damage to the host tissue. Also, dietary antioxidants can reverse the riboflavin-induced protein damage providing an economic and safe anti-ROS therapy.     

Inscuteable-independent apicobasally oriented asymmetric divisions in the Drosophila embryonic CNS

Inscuteable is the founding member of a protein complex localised to the apical cortex of Drosophila neural progenitors that controls their asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterised to date, in both the central and peripheral nervous systems, require inscuteable. Here we examine the generality of this requirement. We show that many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, we focused on the MP2 > dMP2 + vMP2 division. We show that for MP2 divisions, asymmetric localisation and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. We conclude that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable.     

Genetic analysis of a polymorphism in the human apoA-V gene: effect on plasma lipids

Recent discovery and characterization of APOAV suggests a role in metabolism of triglyceride (TG)-rich lipoproteins. Previously, variation at the APOAV locus was shown to modestly influence plasma TGs in normolipidemic samples. The aims of this study were to assess the effects of a polymorphism in APOAV (T-1131C) in terms of its frequency among three dyslipidemic populations and a control population, differences of allele frequency across available ethnic groups, and associations with specific lipoprotein TG and cholesterol compartments. We found a striking elevation in the frequency of the rare allele in a Chinese population (P = 0.0002) compared with Hispanic and European populations. The rare allele of the polymorphism was associated with elevated plasma TG (P = 0.012), VLDL cholesterol (P = 0.0007), and VLDL TG (P = 0.012), LDL TG (P = 0.003), and HDL TG (P = 0.016). Linear regression models predict that possession of the rare allele elevates plasma TG by 21 mg/dl (P = 0.009) and VLDL cholesterol by 8 mg/dl (P = 0.0001), and reduces HDL cholesterol by 2 mg/dl (P = 0.017). The association of the polymorphism with altered lipoprotein profiles was observed in combined hyperlipidemia, hypoalphalipoproteinemia, and hyperalphalipoproteinemia, and in controls. These findings indicate that APOAV is an important determinant of plasma TG and lipoprotein cholesterol, and is potentially a risk factor for cardiovascular disease.